| Wheat is one of the major crops, so its yield and quality are always quite important social issues around the world, especially in China. Yield and quality of wheat are influenced by varieties of environmental conditions, such as drought and salinity. Therefore, enhancement of wheat stress tolerance is critical for its production to cope with the still-increasing adverse environmental situations allover the world. Stress tolerance is a multigenes-controlled phenotype, which may include stress response, ion transport, secondary metabolism and energy flow. Until now, many molecular results regarding stress tolerance mechanisms were achieved using model plants, and have been applied in rice and maize breeding. However, given its hexaploidy and mega-genome, such progress in wheat is very slow, and only a few genes have been reported to improve wheat stress tolerance, which can not reveal the whole truth of its stress tolerance, and are inadequate for wheat breading.Alternatively, Thinopyrum ponticum, the wild relative of wheat with high stress tolerance, is an excellent gene reservoir for wheat breeding. Via somatic hybridization, we bred a wheat introgression line stress tolerance cultivar Shanrong No.3 (SR3) between common wheat stress sensitive cultivar Jinan 177 (JN177) and T. ponticum. Former results showed that some chromatin fragments of T. ponticum intergrated into SR3's genome, and a high frequency of allelic variation and a vast transcriptomic and proteomic change in SR3 happened. These alternations may offer the higher stress tolerance of SR3.Previous study in stress responsible genes TaCHP/TaCEO from SR3 indicated that their ectopic expression increased stress tolerance of Arabidopsis, and influenced the transcription of AtMYB15 in transgenic seedlings. AtMYB15 is one of MYB family members that can improve stress tolerance in Arabidopsis, suggesting MYB family may also play important roles in stress tolerance of wheat. However, little information of MYBs is available in wheat.In this paper, we proposed to identify wheat MYBs based on microarray data of SR3, to analyze their expression pattern under salinity/drought stress and during the whole developmental course, and to compare the variation in their cDNA and genomic sequences between JN177 and SR3, with the aim to primarily characterize the relationship between MYB family and stress tolerance of SR3 and find golden MYB candidates for wheat breeding. The main research contents and results of this work are summarized as follows.1. Wheat MYBs identification from SR3Based on microarray data in our lab,36 MYB unigenes/ESTs were identified using BlastN analysis. The other two MYBs were identified from the SR3 cDNA library in our lab. Totally,38 MYBs with detectable transcription patterns in SR3 at three-leaf stage were identified for further study.2. Expression patterns of MYBs under stressUsing RT-PCR, the express characteristics of 35 MYBs under salinity/drought stress and during the whole development course were analyzed and classified into 42 patterns s in total(3 MYBs were below detectable limit of RT-PCR).MYBs showed differential patterns in response to the same stress. For example, in salinity-stressed SR3 leaves, MYB3/17 showed L1 pattern, a transient response to salinity; while MYB 1/8/9/11/15/16/19/27/31/33 showed L6 pattern, with fluctuant transcription during stress. This indicated that different MYBs play different roles in salinity response.MYBs took different patterns under salinity from those under drought. For instance, in SR3 leaves, MYB31 took L6 pattern under salinity stress, and L41 pattern under drought stress. L41 pattern had a high and a low transcription peak at 0.5h and 12h timepoint, respectively. This indicated that MYB31 plays different roles in salinity and drought response.MYBs showed organ-specific expression patterns. In salinity-stressed SR3, MYB4 showed L11 and L16 patterns in leave and root respectively. L11 pattern is that only mRNA abundance at 3h timepoint was higher than the control. L16 pattern is the transcription that was gradually reduced during stress. This indicated stress response signal transduction chains carry out in an organ-specific manner, and MYBs may function differently in different organs. MYBs had cultivar-specific expression patterns in response to stress. Under salinity stress, MYB3 showed L15 pattern in JN177 roots, whereas L7 pattern in SR3 roots. L15 and L7 patterns are similar to L16 and L6 patterns, respectively. These differences may account for different stress tolerance ability between JN177 and SR3.Apart from the above categories, a few MYBs also acted in the same way under specific conditions. In total, the diverse expression patterns of MYBs suggesting that this family is involved in stress response in a complex way.In order to rule out the relationship between MYBs and the difference in stress tolerance of SR3 from JN177, two analyses were conducted. One is checking SR3-or JN177-specific expression patterns. Among 42 expression patterns, eight (L3/L11/12/13/20/23/37/38) are SR3-specific, and five are JN177-specific. MYBs showing these cultivar-specific expression patterns should be the candidate genes for further study. The other is based the function study of MYB31 (Tamyb31), whose over-expression can improve stress tolerance. MYBs in the same expression patterns as MYB31 are also candidates.Besides, expression patterns of these MYBs at various stage of developmental course in SR3 were checked. The results displayed that MYBs also had their individual expression characteristics during development.3. Sequence variation of MYBsCommon wheat is hexaploid, and SR3 is a somatic hybrid of common wheat. This implies that the RT-PCR product of a certain MYB gene of wheat, especially SR3, may contain more than one transcript. If it is the case, these different transcripts may play different roles in stress response.In order to outline this question, the RT-PCR products and genomic sequence of 15 MYBs were further cloned. Firstly, except for MYB5/22, the other 13 MYBs had more than one transcript in the RT-PCR products. The difference between transcripts concluded two types of diversity sites:Substitution and InDel. The frequency of Substitution was higher than that of InDel. Secondly, distribution of SNP-Indel varied in different MYBs, different alleles. In introns, frequency of SNP-Indel was higher than that in exons; while frequency of SNP-Substitution was a little lower than that in exons. In MYB9/22, Pi value in promoter region was higher than that in coding region. As for Substitutions, frequency of conversion was higher than that of transversion.Above results indicates that SR3 genome suffered a high-frequency of variation. Such sequence variation between JN177 and SR3 may result from two ways, one is the introgression of T. ponticum chromatic fragments, and the other is the genomic shock during somatic hybrid process. SNPs between JN177 and SR3 could cause change in amino acids, so it is quite important to check which transcripts function in stress response in wheat.4. Functional analysis of Tamyb31TaMYB31 was selected as an important candidate gene because of its significant upregulation of transcription under stress in both SR3 and JN177. Its full length cDNA was isolated from cDNA library of SR3, consisting a 192-bp 5'UTR, a 765-bp ORF and a 298-bp 3'UTR. There are SNPs in SR3 and JN177, which led to amino acide variation of the TaMYB31, so TaMYB31 of SR3 was named Tamyb31. Using DNA binding assay and transcriptional activity assay, the transcript with MYB function was identified.RT-PCR and real-time PCR analysis showed that Tamyb31 was induced after 0.5h exposure to salinity/drought stress in SR3. Under treatment with various plant hormones, the expression of Tamyb31 peaked at 0.5h, indicating Tamyb31 may be involved in many signal pathways. Tamyb31 expressed at the whole developmental course, with the highest level at stem jointing stage. Tamyb31 encodes a R2R3 MYB protein with conserved SANT domain when compared with other MYBs; Tamyb31 localizes in nuclei, and binds toâ… /â…¡/â…¡G MYB binding motif.Overexpression Tamyb31 in Arabidopsis increased the tolerance to abiotic stress. Under NaCl, LiCl and KC1 stress, roots of transgenic lines were significantly longer than those of the control, indicating that Tamyb31 can increase the ion stress tolerance of plants. Under drought stress, the overexpression lines also grew better than the control lines. Under mannitol treatment, however, no obvious difference was detected between the overexpression lines and the control lines. This suggests that the enhancement in drought tolerance may be achieved through lowing water loss rate. Under salinity stress, AtP5CS1/AtCBF3 had higher transcripts in the overexpression lines than in the control lines; under non-stressful conditions, AtABF3 had more mRNA abundance in the overexpression lines than in the control lines. Tamyb31 can bind promoter regions of AtABF3/CBF3 using yeast one hybrid assay. These findings suggested that Tamyb31 may confer with abiotic stress through affecting the transcription of these genes.Tamyb31 shared high amino acid sequence similarity with AtMYB15, but their action mechanism may be significantly different from each other. For example, Tamyb31 did not interact with AtICE1, while AtMYB15 did. Tamyb31 had equal binding ability to I/II/IIG MYB motifs, while AtMYB15 prefers to II/IIG motifs.Taken together, in the case of Tamyb31, we conclude that gene-family-ome analysis is a feasible strategy for key functional gene isolation with the aim to stress response mechanism elucidation and transgenic engineer assistant breeding. Besides Tamyb31, other identified MYBs are also worthy of further study.
|
PDF Full Text Request